Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > Molecular Research Topics Forum > Protocols and Methods Forum
Register Search Today's Posts Mark Forums Read

Protocols and Methods Forum Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.

problems with molecular cloning

problems with molecular cloning - Protocols and Methods Forum

problems with molecular cloning - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.

LinkBack Thread Tools Display Modes
Old 03-09-2005, 09:59 AM
lorenzo dep
Posts: n/a
Default problems with molecular cloning

I have a problem an I need urgently some help.
I am cloning a small peptide (300-400 bp) from a CS2 vector(4,1 kb) into an
Huc-int-GFP (about 13 kb).
First I run a PCR to amplify the sequence encoding my fragments and to
introduce the restriction sites for XhOI. I check the PCR products on a
gel, I digest the fragments and the vector Huc-int-GFP with XhOI, I
dephosphorilate the vector with "Shrimp alkalyne phosphatase " and I run a
gel with the fragments and the vector. Then I perform a DNA purification
from the gel (from the right bands) and I run 1 uL on a gel to compare the
relative concentrations of the fragments and the vector.
I do a ligation, I transform E.Coli cells (DH5a),I plate them in petri
disches prepared with LB medium and Ampicilline. After one night at 37 C I
make a "colony PCR" to check which colonies really contain the fragment of
interest and then I chose the positive clones to grow a miniculture in LB
medium and Ampicilline.
Everything "seems" to be fine untill this point.
BUT after all this I make an extraction of the plasmid DNA from the
miniculture, I cut again with XhOI to check if I really have the fragments
that I am cloning.
I run a gel with the digestion product.
What I should see are 2 bands: one for the plasmid (around 13 kb) and the
other for the fragment (between 300 and 400 bp).INSTEAD I see one band
around 2,5 kb and, in some cases, another band abotu 1,6 kb.
Anybody can help me sole this problem?

please write at [Only registered users see links. ]
thank you very much

Lorenzo d'Episcopo
UNDERGRADUATE sudent Biotechnology

Reply With Quote

cloning , molecular , problems

Thread Tools
Display Modes

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off
Trackbacks are On
Pingbacks are On
Refbacks are On

Forum Jump

Similar Threads
Thread Thread Starter Forum Replies Last Post
Found an Ultimate Molecular Cloning Guides online amyjohnson Molecular Cloning Forum 0 11-19-2009 03:42 PM
RA3 Position at Mendel Biotechnology in Molecular Biology & Cloning Mendel Biotechnology Arabidopsis and Plant Biology 0 10-03-2006 05:00 PM
BioInfoMan:reduce your working time on molecular cloning by 80% gaxl Bioinformatics 2 08-15-2006 12:31 AM
problems with molecular cloning lorenzo dep Protocols and Methods Forum 1 03-09-2005 06:04 PM

All times are GMT. The time now is 10:13 PM.

Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2015, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.11559 seconds with 16 queries